The present invention relates to the field of fluorescence analysis and more particularly to an instrument and method for automatically and simultaneously analyzing the excitation and emission spectra of a multicomponent fluorescent sample.
Fluorescence, the emission of electromagnetic radiation in the visible and ultraviolet regions of the spectrum upon the absorption of light of shorter wavelengths, is a useful analytical property of many chemical compounds and biological samples, so that the fluorescence assay has become a universally accepted method for the analysis of a wide variety of substances of interest to chemists, biologists and clinicians. The principal advantages of fluorescence assays are their sensitivity, rapidity and relatively low cost per analysis. The problems or limitations of fluorescence analysis include interference from "blank" fluorescence, Rayleigh and Raman scattering, and an inability to conveniently analyze for several substances simultaneously. Five parameters are available for the characterization of luminescent materials, that is, emission spectrum, excitation spectrum, quantum yield, lifetime and polarization of the emission. While instrumentation exists to obtain all five parameters of luminescence, the acquisition of the data is extremely time-consuming and requires highly trained personnel for both data acquisition and interpretation. As a consequence, in a routine analysis, only a single measurement is generally made, that is, the fluorescence intensity of a sample at a fixed pair of wavelengths of excitation and emission. Present fluorescence spectrophotometers for performing this measurement essentially comprise two monochromaators functioning independently of each other and having two light sources. The output of the first source, generally a Xenon Arc Lamp, passes into the first monochromator (called an excitation monochromator in that it disperses the light) and provides monochromatic light to excite the sample. The excited sample becomes the source for the second monochromator which is called the emission or analyzing monochromator. The collecting optics for the emission monochromator are arranged to detect fluorescence from the sample at an angle other than 180.degree. relative to the exciting light. The angle is usually at 90.degree. or 45.degree. since such an arrangement provides the best rejection of exciting light, and makes it possible to achieve high sensitivity, selectivity and ease of sampling.
In operation, this fluorescence spectrometer can provide either the excitation spectra or emission spectra from a fluorescent sample. Firstly, by adjusting the excitation monochromator to an appropriate excitation wavelength for a given sample and causing the emission monochromator to scan, the emission spectrum of the sample will be presented at the output. Conversely, the emission monochromator can be set to an emission wavelength of the sample and the excitation monochromator may be scanned to produce the excitation spectrum of a sample. Commercially available spectrofluorometers of this type are the Perkin-Elmer MPF-2A, the Aminco-Bowman, the Turner 210, and the Farrand MK-1. All of these instruments use grating monochromators to select the bandwidths of light for excitation and emission. Scanning is accomplished by mechanically displacing the gratings causing the dispersed spectrum to be swept past the monochromator exit slit. The light intensity of each position of the grating is monitored by means of a photomultiplier tube. However, because of the delicate mechanical linkage between the scanning motors and the gratings and the small number of photons emitted, a spectral scan generally takes on the order of 1 to 5 minutes. Thus to obtain 50 fluorescent scans at 50 exciting wavelengths or 50 excitation scans at 50 fluorescent wavelengths could take over 4 hours, a period of time making extraordinary demands on the stability of the instrument if several samples are to be compared.
In contrast, the present invention provides an analytical instrument which can simultaneously measure, in real time, all emission and excitation spectra of a luminescent sample within a given wavelength range and, under favorable conditions, this data can be acquired in the same time as a single measurement of fluorescence intensity in a conventional fluorescence spectrometer. In addition, with a real time dedicated data processor the data may be displayed in a graphical format which is immediately comprehensible to an operator so that a convergence to useful results can be obtained with a minimal waste of computer time. Furthermore, it is possible to record the lifetimes of the emitting species, where the lifetime may be in the range of several hundred picoseconds to tens of seconds, with appropriate exciting sources. In the case of the analysis of multicomponent samples it is possible to obtain (a) the number of emitting species in the sample; (b) the amounts of each species if the spectra of each is known; and (c) a range of possible spectra of the individual components contributing to the total fluorescence, if the spectra are unknown.